Antibodies were first employed in tissue section analysis in 1942 to visualize pneumococcal antigens in organ biopsies from mice infused with live bacteria. Since that time, immunohistochemistry (IHC) has become a mainstay of clinical diagnostics and basic research that is primarily used to assess the spatial distribution of one or two (rarely more) antigens in tissue sections. Despite the high specificity of many antibodies, the concentration of most antigens is insufficient to permit detection by conventional assays without signal amplification. Signal amplification is typically achieved using multivalent, enzyme-linked secondary antibodies that bind the Fc portion of the primary antibody. In bright-field microscopy, the most commonly used enzymatic reporter is horseradish peroxidase, typically used to oxidize 3,3′-diaminobenzidine (DAB), resulting in accumulation of a brown precipitate. Still, the use of secondary antibodies combined with their poor correlation to the primary antigen concentration due to non-linear staining limits both reliable multiplexing and quantitation.
Simultaneous detection of multiple antigens can be subject to additional constraints that limit the utility of existing IHC-based analysis for predictive biomarker development in human clinical trials and clinical diagnostics. Colorimetric detection of four antigens has been reported using multiple enzyme-linked secondary antibodies, but in practice this approach is usually limited to two because of difficulties encountered in sample preparation and imaging. Fluorescent labels can provide a higher signal-to-noise ratio and are more frequently used for simultaneous detection of multiple molecular targets. Practical limitations include the need for primary antibodies generated in dissimilar host species and for non-overlapping reporter emission spectra. This conventional IHC methodology thus does not support the robust generation of multiplexed, quantitative data needed to understand the relationship between tissue microarchitecture and expression at a molecular level.